SUPPLEMENTARY FIGURES A

B

C

D

Figure S1. Genome-wide association study for (A) amylose peak 1 (AM1, DP >1,000), (B) amylose peak 2 (AM2, DP 1000-121), (C) medium chain amylopectin (MCAP, DP 120-37) and (D) short chain amylopectin (SCAP, DP 6-36) as depicted by Manhattan (left) and quantile-quantile (right) plots.

Figure S2. Mapping of the GWAS peak in Chromosome 6 associated with debranched starch structure showing the linkage disequilibrium plot of the most significant SNPs associated with four subfractions of starch corresponding to (A) amylose and (B) amylopectin measured using the SEC method. A scaled section of the chromosome underlying this region is shown where the positions of the genes are labelled with red boxes sized according to their annotation in the Nipponbare reference genome (release 7). The positions of the most significant SNPs are also indicated where the names of the tag SNPs are highlighted in red. The log10-scaled association p-values of these SNPs are shown in the bar plot where bars reflect their relative effect sizes. Also shown are haplotypes formed from the tag SNPs where specific phenotypic ranges for (C) % amylose 1 (AM1), % amylose 2 (AM2), medium chain amylopectin (MCAP) and short chain amylopectin (SCAP) are explained by specific haplotypes.

Figure S3. Targeted haplotyping of waxy gene (LOC_Os06g04200.3) coding for GBSSI showing SNPs detected from the 700K data (McCouch et al., 2016). (A) The structural gene for GBSSI is diagrammatically drawn to scale showing relative location of SNPs detected. (B) Boxplot of the haplotypes detected in a subset of the indica diversity panel (n=61) showing tight association with percent amylose (PAM), amylose 1 (AM1), amylose 2 (AM2), percent amylopectin (PAP), medium chain amylopectin (MCAP) and short chain amylopectin (SCAP). (C) Determination of haplotypic frequency distribution of the different GBSSI haplotypes among the major subpopulations of rice mined from The 3000 Rice Genomes Project (2014).

Figure S4. Mapping of the GWAS peak in Chromosome 7 associated with amylose 1 (AM1) and (SCAP) fractions. (A) The linkage disequilibrium blocks formed by the 34 significantly associated SNPs at the genetic interval flanked by LOC_Os07g11020 and LOC_Os07g11670 structural genes (shown as red horizontal bars). These SNPs are based on the Nipponbare reference genome (release 7). Also shown together with the SNP IDs are the alternative alleles. SNP IDs highlighted in red are the 12 tag SNPs from which the haplotypes were formed (B). The bar chart shows the log10 of the association p-values of the SNPs, and the relative thickness of the bars reflects the effect size of each SNP. The bars colored black and the bars colored red mean that these SNPs have positive and negative allelic effects, respectively. The haplotype distribution in the 3000 rice genomes is shown (C).

Figure S5. Targeted haplotyping for gene coding for bHLH transcription factor (LOC_Os07g11020) showing tagged SNPs detected from the 700K SNPs data (McCouch et al., 2016). (A) The structural gene is diagrammatically drawn to scale showing relative location of SNPs detected. (B) Boxplot of the haplotypes detected in a subset of the indica diversity panel (n=30) showing tight association with percent amylose (PAM), amylose 1 (AM1), amylose 2 (AM2), percent amylopectin (PAP), medium chain amylopectin (MCAP) and short chain amylopectin (SCAP). (C) Determination of haplotypic frequency distribution of the different bHLH haplotypes among the major subpopulations of rice mined from The 3000 Rice Genomes Project (2014). (D) The haplotype based on most significant SNP (snp_07_6067391) located in exon 5 of bHLH demonstrating strong influence on percent amylose (PAM) due to amylose 1 (AM1) and percent amylopectin (PAP) due to short chain amylopectin (SCAP) levels.

A

B

Figure S6. Determination of correlation of starch hydrolysis kinetics (A) and k-value estimations (B) of lines carrying different haplotypes using IR36 amylose extender (IR36ae) mutant as less digestible control.

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0 50 100 150 200

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tarc

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lyze

d

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IR36ae

Haplotype 1

Haplotype 8

Haplotype 4

SUPPLEMENTAL TABLES Table S1. Amylose classification of indica diversity panel using iodine and SEC estimation methods.

Amylose Class Range Iodine SEC

Waxy 0-2.0 6 2

Low 2.1-19.9 43 165

Intermediate 20.0-24.9 117 49

High 25.0-30.0 78 28

Total

244 244

Table S2. List of most significant (MS) and linked (tag) SNPs detected in Chromosome 6 and 7 and their significance and

allelic effects based on 6 fractions defined for debranched SEC. Significance is measured using –log10(p) value while

allele effects is measured using beta-coefficient. Three SNPs in GBSSI (LOC_Os06g04200) were added as reference for

Chromosome 6, with the location of the MS included based on GBSI start codon in kilobasepairs (Kbp).

(See Table S2.xlsx)

Table S3. Novel SNPs identified at the structural gene of Granule Bound Starch Synthase I using a subset of the 700K SNPs data.

SNP Name Location SNP

Location Type of

Amino Acid Change Exon:

Amino Acid Change

% Amylose

Ref Alt R-sq SE p-value -log10(p)

snp_06_1766647 1766647 A C 5’-UTR N/A N/A 0.0443 1.9215 0.0566 1.2475

snp_06_1767387 1767387 G A exonic synonymous SNV Exon3: R118R 0.0182 4.8109 0.1515 0.8195

snp_06_1768006 1768006 A C exonic nonsynonymous SNV Exon5 or Exon6: Y224S 0.0504 2.8292 0.0452 1.3445

snp_06_1768724 1768724 T C exonic synonymous SNV Exon 8 or Exon9: P362P 0.0735 2.6111 0.0196 1.7085

snp_06_1768998 1768998 C T exonic nonsynonymous SNV Exon10: P415S -0.0225 6.9040 0.9253 0.0337

snp_06_1769525 1769525 G A intronic N/A N/A -0.0059 4.8697 0.4249 0.3718

snp_06_1769710 1769710 A G intronic N/A N/A -0.0148 1.9097 0.7233 0.1407

snp_06_1769727 1769727 A T intronic N/A N/A -0.0114 4.0210 0.5706 0.2437

Table S4. SNPs and other structural variations in GBSSI previously reported in literature. The major allelic variations are

shaded in gray.

Mutation Genotype and Phenotype Effects Publication

(CT)n Repeat at 5’-UTR of Waxy exon 1

Also known as RM190, possibly a closely-linked marker rather than the causal mutation for AAC variation

Bligh et al. (1995) Ayres et al. (1997) Bergman et al. (2001)

23-bp duplication at Exon 2 Present only in waxy varieties, 23 bp duplication 100 bp downstream the ATG causing a premature stop codon which inactivates the Waxy gene wx = null frameshift mutation coding non-functional GBSSI leading to glutinous rice grain

Wanchana et al. (2003) Mikami et al. (2008) Fitzgerald et al. (2009)

Exon 2 A/G SNP Wxhp = Low AAC Yunnan landraces A → G Exon 2 at position +497 Asp → Gly substitution leading to reduction in GBSSI activity

Liu et al. (2009)

Exon1-Intron 1 Boundary G/T SNP* Wxa = G allele = intermediate to high amylose (indica) Wxb = T allele = glutinous to low amylose (japonica) Only distinguishes glutinous and low amylose types from those with intermediate and high amylose content AGGT → AGTT SNP Reduction in pre-mRNA splicing efficiency and promotion of alternative splicing at cryptic sites in exon 1 = decreased production of functional enzymes = glutinous and low amylose phenotypes

Wang et al. (1995)

Ayres et al. (1997) Bligh et al. (1998) Cai et al. (1998) Isshiki et al. (1998) Larkin and Park (1999)

Exon 4 A/G SNP A → G at +715 Asp → Gly Wxop = indica varieties from India, Nepal, Indonesia and China with opaque and chalky endosperm with very low amylose content

Larkin and Park (1999) Mikami et al. (1999)

Exon 4 G/A and Exon 5 T/C SNP Wxmq = low AAC rice cv. Milky Queen Two base changes within the coding region:

Sato et al. (2002)

G → A Exon 4 T → C Exon 5 Missense amino acid substitution

Exon 6 A/C SNP* Wxin = discriminates intermediate from high amylose Non-conservative mutation at +1,083 Tyr → Ser substitution at the active site of the enzyme reducing its specific activity

Accessions belonging to aromatic group and tropical japonica with intermediate AAC

Non-conservative, changes polarity and function of enzyme

Impacts enzyme activity and amylose content

Cai et al. (1998) Larkin and Park (2003) Chen et al. (2008)

Dobo et al. (2010) Kharabian-Masouleh et al. (2011)

Exon 9 T/C Silent mutation (does not change amino acid sequence)

Exon 10 C/T SNP* Proline → Serine Distinguishes high amylose

Larkin and Park (2003) Dobo et al. (2010)

*Most significant impact on starch (Cai et al., 1998)

Table S5. Identification of putative cis-regulatory elements binding to the 5’-promoter region of the structural gene of GBSSI using online JASPAR 2016 Plant database search tool (Mathelier et al., 2016). 100% relative profile score threshold was used for the search.

Model ID Model name Score Relative score Start End Strand Predicted Site

Sequence

MA1049.1, MA1034.1 ERF094, Os05g0497200 13.294, 14.061 1.000006908, 1.000006685

176 183 -1 CGCCGCCG

MA1053.1, MA0980.1,MA1052.1 ERF109,RAP2-10, RAP2-6 14.122,14.349,14.712 0.999992949, 0.999990699, 1.000001819

177 184 -1 GCGCCGCC

MA0121.1 ARR10 9.98 1.00001178 214 221 1 AGATCCGC

MA1063.1,MA1019.1, MA1066.1

TCP19,Glyma19g26560.1, TCP23 14.648, 14.509, 15.171

1.000002482, 0.9999955, 1.0000007

333 342 1 TGGGGCCCAC

MA1019.1,MA1063.1,MA1066.1 Glyma19g26560.1,TCP19,TCP23 14.509,14.648, 15.171

0.999995573, 1.000002482, 1.00000072

343 351 -1 GGGGCCCAC

MA0053.1, MA0064.1 MNB1A, PBF 8.115, 8.062 1.000019324, 0.999992556

678 682 -1 AAAGC

MA0053.1, MA0064.1 MNB1A, PBF 8.115, 8.062 1.000019324, 0.999992556

901 905 1 AAAGC

MA0959.1, MA0959.1, MA0958.1 AIB, BHLH13 14.865, 14.188 1.000000943, 0.999996191

1444 1451 -1, 1 GCACGTGC

MA0020.1, MA0053.1, MA0064.1 Dof2,MNB1A, PBF 8.76, 8.115, 8.062

0.999993793, 1.000019324, 0.999992556

1547 1552 1 AAAGCA

MA0020.1 Dof2 8.76 0.999993793 1809 1814 -1 AAAGCA

MA0053.1 MNB1A

8.115 1.000019324 1810 1814 -1 AAAGC

MA0064.1 PBF 8.062 0.999992556 1810 1814 -1 AAAGC

Table S6. Motif analysis for starch-related genes and bHLH done using locally installed Meme package (Bailey et al.,

2009) with Jaspar Plant database (Mathelier et al., 2016). Those motifs were not showing any hits with the local version

of Jaspar database was removed from the table.

(See Table S6.xlsx)

Table S7. List of differentially-expressed genes identified from contrasting lines of Haplotype 8 (high amylose) and

Haplotype 4 (waxy).

(See Table S7.xlsx)

Supplementary Table 8. The turquoise module showing the most distinct expression pattern consisting of 441 nodes which

codes for most of the genes of interest.

(See Table S8.xlsx)

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